Purified lactase

ABSTRACT

The present invention relates to a lactase solution comprising a lactase solution comprising less than 10 g/kg of poly and oligosaccharides, a process for the production of such a lactase solution from an untreated lactase solution, a sterilized lactase, whereby such lactose is filter sterilized in-line with the milk production process.

This application is a continuation of U.S. application Ser. No.10/474,113 filed Oct. 6, 2003 (issued as U.S. Pat. No. 7,955,831 on Jun.7, 2011), which is the U.S. national phase of international applicationPCT/EP02/03680 filed on Apr. 3, 2002, which designated the US and claimspriority to EP Application No. 01000102.2, filed Apr. 4, 2001. Theentire contents of these applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to purified lactase and the productionthereof.

BACKGROUND OF THE INVENTION

Lactase or beta-galactosidase (EC: 3.2.1.23) is an enzyme, which canconvert lactose (disaccharides) into the monosaccharides glucose andgalactose. Lactose is present in dairy products and more particularly inmilk, skimmed milk, cream and other milk products. The breakdown oflactose occurs in the intestinal wall of the human body (and in othermammals) by the natural presence of lactase.

Many humans (and other mammals) suffer from lactose-intolerance, whereinlactase is absent or partially absent in their digestive system. In casewhere lactose is part of the food or feed, decreased digestion oflactose may lead to intestinal trouble.

Nowadays lactase is added to milk to breakdown the lactose present.Lactase may be added to milk either before or after pasteurisation orsterilization. In general lactase will be inactivated duringpasteurisation or sterilization treatment. When lactase is added beforesterilization a large amount of lactase may be required in order toreduce the storage time between addition andpasteurisation/sterilization. Although lactase is an active enzyme onehas to keep in mind that milk is processed and stored generally attemperatures between 0 and 8° C.

The other possibility is the addition of the enzyme after pasteurisationor sterilization of the milk and before packing. In this case lactasemay be added in a lower amount, as it may be at least 10 to 24 hoursbefore the milk is consumed. The enzyme can digest lactose, which may bepresent during transport and storage in the factory, shop and in therefrigerator of the consumer.

There are several ways to sterilize lactase, for example by chemicaland/or heat treatment. However, because of its application in food orfeed, sterile filtration is a preferred option.

In the journal Voedingsmiddelentechnologie 13 (1980), 23, a method,which is also described in British patent specification 1477087, isfurther illustrated. Lactase, usually used by the dairy processingindustry as an aqueous solution to which one or more stabilizing agents,such as glycerol, can be added, is filtered before use. The filteredenzyme solution is pumped through a sterile filter then injected via adosing device into a production line of previously sterilized orpasteurised milk and then mixed with the milk which is subsequentlypacked under aseptic conditions in uniform packs.

However, in practice, the sterile filter often blocks due to degradedprotein, poly- and oligosaccharides remaining in the enzyme solutiondespite filtering. According to EP 145092, such degradation generallyincreases the longer the enzyme is stored prior use and may be promotedby the considerable period of time between the production of the enzymeand its use in the dairy processing industry. The repeated cleaning orreplacement of the sterile filters is not an option since stopping thewhole process requires sterilisation before starting again.

EP 145092 describes a process for the sterile filtration of lactasewithin 14 days of it being produced. EP 145092 describes that lactaseshould be sterile filtered after recovery and purification of lactaseproduced by fermentation, but before the formation of degradationproducts which are sufficient to clog the sterile filter. However theapproach of sterile filtering freshly produced lactase solution does notfulfil the need of lactase solution which can be filtered in-line andwhich can be added to the sterilized/pasteurised milk. The lactasedescribed in EP 145092 is derived from the yeast Kluyveromyces that isused widely in the dairy industry. The polysaccharides are probablyparts of host cell walls, which are formed during the recovery process.

SUMMARY OF THE INVENTION

The present invention provides a lactase solution comprising less than10 g/kg of poly and oligosaccharides.

The present invention also provides a process for the production of alactase solution, whereby the poly and oligosaccharides present in anuntreated solution are separated from the lactase solution.

The present invention also provides a process for producing lactasecontaining milk whereby the lactase is sterilized before the lactase isadded to the milk.

The present invention also provides a sterilized lactase solution andalso dairy products comprising the sterilized lactase solution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to purified lactase and in particular tonovel processes for the production of lactase free from poly andoligosaccharides. The removal of poly and oligosaccharides allows easierfilter sterilization of the lactase solution since the filter does notbecome blocked with poly and oligosaccharides. As a result, purifiedlactase can be filter sterilized “in line” with a milk productionprocess, thereby negating the need for such a filter to be repeatedlyunblocked.

The present invention provides a solution of lactase, which can bestored and which, after storage, is still free of clogging compoundssuch as poly and or oligo saccharides. Preferably this solution can bestored after recovery and purification from the fermentation process forat least 15 days, preferably more than 30 days and more preferably morethan 120 days.

Free of poly- and oligosaccharides means that less than 10 g/kg of poly-and oligosaccharides are present in the lactase solution. Preferablyless than 5 g/kg, more preferably less than 2 g/kg and most preferablyless than 1 g/kg of poly- and oligosaccharides are present in thelactase solution.

The lactase solution of the present invention is very suitably ofsterile filtration, since the low concentration of compounds such aspoly- and oligosaccharides, allows the lactase solution to be sterilefiltered with less chance of clogging the filter. The filter can be usedfor a long time preferably at least four times longer than in case ofuse of lactase solution, which is not substantially free ofpolysaccharides.

The lactase solution is preferably an aqueous solution of lactase. Thelactase solution in general will contain from 10 to 100000 NLU/g,preferably from 100 to 10000 NLU/g.

The lactase solution may comprise one or more solvents or otheradditives, which bring the enzyme activity to the desired level and mayfurther stabilize the enzyme. Suitable solvents are, for example,sorbitol and glycerol. These solvents may be added to a concentration offrom 10 to 70 w/w %, or more preferably 30 to 70% w/w, of the lactasesolution. Suitable additives, which stabilize the enzyme, are forexample, hydrolysed lactose, glucose, mannitol and salt buffers.According to one embodiment of the present invention the lactasesolution, which is generally free of clogging compounds such aspolysaccharides, can be obtained by purifying an untreated lactasesolution in a chromatographic process whereby all the compoundsresponsible for clogging a filter are separated from the lactasesolution.

Even in cases where the untreated lactase solution is stored for atleast 15 days or even more than 30 days before the chromatographicprocess, the purified lactase solution can still be easily sterilefiltered without the filter becoming clogged.

Unexpectedly, the use of chromatography makes it possible to remove all(poly- and oligosaccharides, proteins, peptides etc.) compounds, whichmight clog the sterile filter. Surprisingly, it was found that only onechromatographic step was required to remove all polysaccharides and allother unknown non-protein compounds, which are responsible for cloggingthe sterile filter. One has to keep in mind that the lactase solutionalthough being recovered and purified from the fermentation broth, willcontain at least polysaccharides, which are at least partly convertedinto degradation products. Typical concentration of polysaccharides inuntreated lactase solutions are from 10 to 100 g/kg.

Commercial lactase preparations may contain from 40 to 60% w/w ofglycerol. The viscosity of the lactase preparation is therefore expectedto be related to the amount of glycerol present. However we have foundthe viscosity of a lactase preparation can be reduced significantly bythe removal of polysaccharides from the lactase preparation. Thisreduction of viscosity makes it possible to pass the lactase preparationthrough the sterile filter at a reduced pressure difference or atsimilar pressure difference on the filter, to allow more lactasesolution to pass through the filter compared to a lactase solution notpurified according to the present process. The invention provideslactase solutions comprising from 10 to 70 w/w % of glycerol, preferably30 to 70 w/w % and more preferably 40 to 70 w/w % of glycerol having aviscosity of less than 100 mPa, preferably less than 80 mPa and evenmore preferably less than 60 mPa.

Separation of the clogging compounds from the lactase solution can beachieved by binding of the lactase to an appropriate chromatographicresin. Suitable resins which can be used to separate clogging compoundsfrom a lactase solution are for instance anion and cation exchangeresins. Anion exchangers, for instance Q-sepharose, can be used when alactase solution at a pH above its isoelectric point is applied to ananion exchanger equilibrated in the same pH. Lactase, but not cloggingcomponents, is bound to the resin. The bound lactase can be eluted(desorbed), free of clogging compounds, from the resin by increasing theionic strength and/or changing the pH of the anion exchange resin. Thechange in ionic strength and/or pH during desorption or elution can takeplace under a stepwise or continuous gradient.

The same separation can be achieved with a cation exchanger, forinstance SP-sepharose, wherein the lactase preparation is applied to theresin below its isoelectric point. We have found that HAP(hydroxyapatite) chromatography does not give separation of the poly andoligosaccharides from the lactase. A possible explanation might be thatpoly and oligosaccharides are also bound to the HAP matrix.

Preferred resins are hydrophobic interaction media (HIC). On a HICmedia, separation can be obtained based on the differences inhydrophobicity. Different HIC resins are available which containdifferent ligands, for instance ethyl, propyl, butyl, phenyl and octyl.By applying an aqueous lactase solution under conditions which, permitbinding of lactase to the resin, it is possible to separate lactase fromthe clogging compounds.

A typical protocol for promoting binding or adsorption to a HIC resin isapplying an aqueous lactase solution under non-denaturing pH and arelatively high ionic strength to the resin. The high ionic strength canbe obtained by adding salts to the lactase solution. Appropriate saltsare for instance ammonium sulphate, sodium chloride and sodium sulphate.

After the binding or adsorption step, lactase can be eluted or desorbedby decreasing the ionic strength. The change in ionic strength and/or pHduring elution or desorption can take place under a stepwise orcontinuous gradient. Other suitable resins are for instancegelfiltration media and hydrophobic charge induction media (combinationof HIC and ion exchange chromatography).

The lactase provided in the present invention produced by a yeast,preferably a Kluyveromyces strain more preferably K. lactis or K.fragilis. Such lactase is purified according to the invention resultingin a lactase solution which is easy to sterilize. The removal of allcompounds responsible for clogging the sterile filter is very surprisingbecause after the fermentation process, the lactase, which is beingrecovered and purified, may contain polysaccharides, which are at leastpartly converted into degradation products. Therefore it is believedthat protease and other contaminating proteins also present in lactasesolution are removed in the chromatography step. The sterile filterpreferably used to sterilize the lactase solution is in general presentin the milk in-line production process. The sterile filtering treatmentis preferably carried out in-line with respect to the milk productionprocess, whereby one or more membrane filters are used. A suitablesterile filter is for example a membrane filter having a pore size of0.22 μm.

The lactase solution according to the invention is advantageously usedin the preparation of pasteurised milk.

EXAMPLE 1

The example describes a typical chromatographic purification procedureof lactase using phenyl sepharose LS as chromatographic resin.

The commercial product Maxilact® 5000 LX (obtainable from DSM, TheNetherlands) containing 5000 NLU/g was diluted 5 times withdemineralised water, ammonium sulphate is added to a final concentrationof 1 M after which the pH was corrected to 7.5.

A 20 ml sample was applied to a 20 ml HiPrep phenyl 16/10 column havinga diameter of 16 mm and length of 10 cm, at a linear flow rate of 150cm/h. The column was equilibrated with 1 M ammonium sulphate in 100 mMTris pH 7.5. After loading the column was washed with equilibrationbuffer at a flow rate of 150 cm/h until the baseline was reached.Elution of lactase was done under a step gradient at 150 cm/h (100 mMTris pH 7.5). After elution of the lactase the lactase solution wasdesalted and concentrated to a final activity of approximately 10.000NLU/g. After concentration the lactase solution was formulated withglycerol at a final concentration of 50% w/w, the final lactase activityafter formulation is approximately 5.000 NLU/g.

Lactase activity was determined by the hydrolysis of the substrateo-nitrophenyl-β-galactopyranoside (ONPG) into o-nitrophenyl andgalactose. The reaction was terminated by the addition of sodiumcarbonate. The absorbance of the o-Nitrophenyl formed, being yellow inalkaline medium, was used to measure the activity of the enzyme(expresses as NLU/g). The procedure is published in the. FCC, fourthedition, Jul. 1, 1996, page 801 to 802/Lactase (neutral)(β-galactosidase) activity

The results are shown in the Table 1

TABLE 1 g/kg sugars Poly- and Activity before after oligosaccharidesSample NLU/g inversion inversion g/kg Maxilact LX 5000 5000 1 57.6 56.6Purified Lactase 5000 <1 2.21 <2

Poly- and oligosaccharide content was determined by measuring the amountof free sugar and the amount of sugar present after the acid inversionof the polysaccharides.

The polysaccharide contents were determined by means of High PerformanceLiquid Chromatography (HPLC). The detection was performed using a RI(refraction index)-detector. The column used was a BioRad Aminex HPX87N, length 30 cm, inner diameter 7.8 mm, thermostated at 85° C. Themobile phase was a solution of 0.71 g sodium sulphate in 1 liter waterat a flow rate of 0.68 ml per minute.

Two different samples pre-treatment were performed, both with andwithout acid inversion. The sample pre-treatment without inversion wasdone by weighing 5 g sample in a volumetric flask of 50 ml, dissolvingin mobile phase and injecting 5 μl onto the column.

The sample pre-treatment with inversion was done by weighing 2 g samplein a centrifuge tube, adding 3.00 ml water and 2.50 ml hydrochloric acid2.58 mol/l, heating for 75 minutes at 100° C. and adding 2.50 ml sodiumhydroxide 2.58 mol/l. 5 μl of the resulting solution was injected ontothe column.

The glucose content was calculated using a standard solution with aconcentration of 400 mg glucose in 50 ml mobile phase. Theconcentrations of trisaccharide, disaccharide and fructose werecalculated using a response factor, relative to glucose.

EXAMPLE 2

The example describes the sterile filtration of lactase solutions.

A syringe was filled with 1 ml Maxilact LX 5000, and a sterile filter,Millex GV 0.22 μm from Millipore with a surface of 4.91 cm², was placedon top of the syringe. After applying hand pressure it was not possibleto filter the product through the filter, increasing the pressure causedthe filter to break.

Another syringe was filled with 1 ml of purified lactase formulated with50% w/w glycerol (end concentration), prepared as described in Example1, and a sterile filter, Millex GV 0.22 μm from Millipore with a surfaceof 4.91 cm², was placed on top of the syringe. After applying handpressure it was surprisingly easy to sterile filter at least 1 ml of thelactase solution through the filter.

Therefore, the use of chromatography allowed all compounds which mayhave clogged the sterile filter to be removed, and as a result sterilefiltration was not problematic.

EXAMPLE 3

The example describes viscosity measurements of formulated lactasesolutions.

The viscosity of both the commercially available Maxilact LX 5000 and ofthe purified lactase formulated with 50% w/w glycerol (endconcentration), prepared as described in Example 1 was measured.Commercially available Maxilact also contains 50% w/w glycerol.

The viscosity was measured with a Physica UDS 200 at 25° C., using a MK21 cone probe.

Table 2 shows the viscosity of the purified lactase formulated at 5.000NLU/g with 50% glycerol (end concentration) significantly drops as aresult of the purification in which all clogging compounds are removed.

TABLE 2 Results of viscosity measurements Viscosity mPa Product shearrate = 100 (s − 1) Commercial Maxilact LX 5000 170 Purified lactaseformulated  40 with 50% glycerol

The invention claimed is:
 1. A lactase solution comprising Kluyveromyceslactase and a stabilizing solvent, wherein the solution comprises lessthan 10 g/kg of poly and oligosaccharides, and wherein the stabilizingsolvent is sorbitol or glycerol.
 2. The lactase solution according toclaim 1 which is storage stable for at least 15 days.
 3. The lactasesolution according to claim 1, which is an aqueous solution.
 4. Thelactase solution according to claim 1 further comprising at least onestabilizing additive.
 5. The lactase solution according to claim 4,wherein the additive is selected from the group consisting of hydrolysedlactose, glucose, mannitol and salt buffers.
 6. The lactase solutionaccording to claim 1, wherein lactase is contained in an amount of from10 to 100,000 NLU/g.
 7. The lactase solution of claim 1, wherein theKluyveromyces lactase is a K. lactis lactase or a K. fragilis lactase.8. The lactase solution of claim 1 comprising from 10 to 70 w/w %glycerol and having a viscosity of less than 80 mPa·s.
 9. The lactasesolution of claim 1, wherein the lactase solution is used to breakdownlactose present in milk.
 10. A process for the production of the lactasesolution according to claim 1, comprising: providing an untreatedsolution of lactase; separating poly and oligosaccharides from thelactase solution; and adding a stabilizing solvent to the separatedlactase solution.
 11. The process according to claim 10, wherein theseparating step is accomplished by using only one chromatographic step.12. A sterilized lactase solution, produced by a process comprisingsterilizing the lactase solution of claim 1 on a sterile filter.
 13. Adairy product comprising the sterilized lactase solution according toclaim
 12. 14. A process for producing sterilized lactase, comprising:providing the lactase solution according to claim 1; and sterilizingsaid lactase solution with a sterile filter.
 15. A process for producinglactase-containing milk, comprising: sterilizing the lactase solutionaccording to claim 1; and adding said lactase solution to milk.
 16. Theprocess according to claim 15, wherein the sterilization is carried outon at least one filter positioned in line with the milk productionprocess.
 17. The process according to claim 15, whereby the milk ispasteurised or sterilized milk.